Transcriptome Analysis of Campylobacter jejuni and Campylobacter coli during Cold Stress

Campylobacter spp. are known to cause campylobacteriosis, a bacterial disease that remains a public health threat. Campylobacter spp. are prevalent in retail meat and liver products, and the prolonged survival of Campylobacter in the low temperatures needed for storage is a challenge for food safety. In this study, RNA-seq was used for the analysis of the C. coli HC2-48 (Cc48) and C. jejuni OD2-67 (Cj67) transcriptomes at 4 °C in a nutrient-rich medium (chicken juice, CJ) and Mueller–Hinton broth (MHB) for 0 h, 0.5 h, 24 h and 48 h. Differentially expressed genes (DEGs) involved in flagellar assembly were highly impacted by low temperatures (4 °C) in C. coli HC2-48, whereas genes related to the ribosome and ribonucleoprotein complex were modulated for C. jejuni OD2-67 at 4 °C. Most of the DEGs in cells grown at 4 °C in the two medium formulations were not significantly expressed at different incubation times. Although more DEGs were observed in CJ as compared to MHB in both Campylobacter strains, the absence of common genes expressed at all incubation times indicates that the food matrix environment is not the sole determinant of differential expression in Campylobacter spp. at low temperatures.


Introduction
Campylobacter spp. are microaerobic, thermophilic bacteria (optimal temperaturẽ 42 • C) that lack cold-shock-response genes; despite this limitation, Campylobacter strains survive at the low temperatures used for food storage conditions [1]. The microenvironment has a profound influence on the survival of Campylobacter [2]; for example, a nutrient-rich environment containing meat or liver juice facilitates the survival of Campylobacter strains at low temperatures and may contribute to the high frequency of Campylobacter spp. in retail meats and liver products during slaughter, processing and storage [1,3]. We previously reported the high prevalence of C. jejuni and C. coli strains in retail liver, chicken, pig and beef products [4][5][6]. Aerotolerance and co-contaminants such as Staphylococcus aureus also improve Campylobacter survival in adverse environments including aerobic conditions and low temperatures [7,8].
A prior study reported that Campylobacter survival at low temperatures is presumably an active process where changes occur in lipids, oligosaccharides and polysaccharides [9]. In contrast, another report suggested that adaptation to low temperatures is a passive mechanism where various genes (e.g., clpB, trxC, perR) and two-component regulatory systems (RacRS) were essential for survival in a nutrient-rich or minimal medium [10]. Other genes that might contribute to Campylobacter survival at low temperatures include sodB, luxS and genes related to motility, chemotaxis, energy production/conversion, amino acid transport/metabolism and lipid transport/metabolism [9,[11][12][13]. Furthermore, the acquisition of cryoprotectant molecules might also contribute to survival at low temperatures [10,13].
It is well-established that microorganisms cope with environmental adversity by altering gene expression. Global changes in Campylobacter gene expression were influenced by medium,

Preparation of Cells for RNA Isolation
Bacterial cells (log phase cultures) were pelleted at 6000 rpm for 10 min and suspended in freshly prepared MHB to an OD 600~0 .5. Prepared bacterial suspensions were maintained at 42 • C in microaerobic conditions for 2 h. For control samples at 42 • C, Cc48_42 (C. coli HC2-48) and Cj67_42 (C. jejuni OD2-67), cells (150 µL bacterial suspensions) were collected from cultures incubated at 42 • C in microaerobic condition. Control samples were then immediately mixed with TRI reagent (Zymo Research, Irvine, CA, USA) (700 µL) and maintained at −70 • C until RNA was isolated.

RNA Extraction
Frozen samples were allowed to reach room temperature, and cells were disrupted with vigorous vortexing. Total RNA was extracted with the Directzol RNA isolation kit (Zymo Research, Irvine, CA, USA) and TRI reagent as recommended by the manufacturer. On-column DNA digestion was performed using instructions provided with the Directzol RNA isolation kit followed by an additional two-step DNase treatment (TURBO DNA-free Kit, Invitrogen, Vilnius, Lithuania) to eliminate any contaminating DNA. Total RNA was quantified with the Nanodrop spectrophotometer and the Qubit™ RNA HS Assay Kit (Invitrogen, Eugene, OR, USA). Absence of genomic DNA contamination in RNA samples was confirmed by PCR with primers of housekeeping genes glyA and aspA as described previously [7]. The quality of RNA was checked by denaturing RNA electrophoresis in agarose gels [18]. RNA samples (5-10 µg) were treated with the Ribominus Transcriptome Isolation Kit (bacteria) (Invitrogen, Carlsbad, CA, USA) for removal of ribosomal RNA and re-analyzed by denaturing RNA electrophoresis. Prior to preparation of cDNA libraries, triplicate samples from replicated biological experiments were mixed together in equal concentration.

cDNA Library Preparation, Sequencing and Expression Analysis
Approximately 100 ng of purified mRNA samples were used as starting material for cDNA libraries using the Illumina TrueSeq Stranded mRNA Library Prep kit as recommended (Illumina, San Diego, CA, USA) with minor modifications. The procedure for selective enrichment of mRNA was skipped, and cDNA libraries were quantified with the Qubit dsDNA HS Assay kit. The quality of cDNA libraries was determined using the Agilent High Sensitivity DNA kit and Agilent Bioanalyzer 2100 (Agilent, Santa Clara, CA, USA). Prepared cDNA libraries were sent to service provider (Quick Biology Inc., Monrovia, CA, USA) for sequencing in the Illumina HiSeq 4000 platform with a read length of 2 × 150 bp (paired-end run). All the original sequence reads for this experiment were deposited in GenBank and are accessible in the Sequence Read Archive (SRA) database within Bio project id: PRJNA828109.
RNA sequences were analyzed with the CLC Genomics Workbench v. 20 as described previously [19]. Raw sequence reads were trimmed to remove adapter sequences and ambiguous reads as needed, and all reads <10 bp were discarded. Genomic sequences of C. jejuni OD2-67 (NZ_CP014744, NZ_CP014745 and NZ_CP014746) and C. coli HC2-48 (NZ_CP013034 and NZ_CP013035) were downloaded from Ref Seq (https://www.ncbi.nlm.nih.gov/refseq, accessed on 9 March 2020) and used as references. Sequence reads that mapped to rRNAs from bacteria and chicken were removed from downstream analyses. Read counts were normalized and differential expression was conducted in the CLC Genomic Workbench v. 20 with default settings. All noncoding sequences, pseudogenes, and frameshifted genes were excluded from analysis. Differentially expressed genes (DEGs) with fold-change values ≥ 1.5 or ≤−1.5 and false discovery rates (FDR) < 0.05 were considered significant. Heatmaps were created using the ComplexHeatmap package (Bioconductor), and Venn diagrams were created with Venny 2.1 (https://bioinfogp.cnb.csic.es/tools/venny, accessed on 9 March 2020). Genome-wide functional annotation of C. jejuni OD2-67 and C. coli HC2-48 was carried out with the noncurated eggNOG v. 5.0 database [20] (http://eggnog-mapper.embl.de, accessed on 9 March 2020) with e-value of 0.001, seed ortholog score of 80 and both query and subject cover cutoff value of 60%. Gene annotation and gene enrichment analysis for common DEGs were conducted using STRING v. 11 (https://string-db.org, accessed on 9 March 2020) with default settings. Schematic representations of expression levels in different functional groups were executed with the Circos Table viewer (http://mkweb.bcgsc.ca/tableviewer, accessed on 9 March 2020).
Nucleotide sequence similarity analyses (BlastN, BLAST Atlas) were conducted with genomes of C. coli HC2-48 and C. jejuni OD2-67 using files retrieved from RefSeq in the Gview server (https://server.gview.ca, accessed on 9 March 2020); the settings included e-values of 1e-10, alignment length cutoff value of 100 bp and identity cutoff values of 80%. Other genes with similar names and functions were included as orthologous genes.

Validation of Differential Gene Expression by qRT-PCR
RNA samples from biological experiments (triplicates) were extracted as described above for RNA isolation. The primers used for target genes and endogenous controls (ilvC and slyD) are listed in Table S2. Quantitative real-time PCR (qRT-PCR) was conducted using the QuantiTect SYBR Green RT-PCR kit (Qiagen) in MicroAmp Fast 96-well reaction plates (Applied Biosystems) as described in [7]. One-step qRT-PCR cycles were executed using the StepOne Real-Time PCR System (Applied Biosystems). Relative quantification of gene expression was conducted using the 2 −∆∆Ct method [21], and statistical analysis was performed using GraphPad Prism v. 9 (https://www.graphpad.com/scientific-software/ prism, accessed on 9 March 2020).
Six genes were significantly upregulated in MHB but not in CJ at all incubation times. These DEGs included the following: lptD, LPS assembly protein; tolA, TonB C-terminal domain-containing protein; murE, UDP-N-acetylmuramoyl-L-alanyl-D-glutamate-2, 6diaminopimelate ligase; AR446_RS05150, hypothetical protein; AR446_RS04310, DUF342domain-containing protein; and lptA, LPS periplasmic transport protein. Two genes, rpmL (50S ribosomal protein L35) and shlA (filamentous hemagglutinin N-terminal-domaincontaining protein) were downregulated in MHB but not in CJ. However, no genes were identified that were significantly expressed in CJ but not in MHB.
C. jejuni OD2-67: Fifty eight genes showed significant differential expression in MHB and CJ at 4 • C vs. 42 • C in all incubation times (Tables 3 and S6). In MHB, 195 genes were differentially expressed at 4 • C vs. 42 • C in all incubation times (Tables S7 and S8), and 71 genes were significantly expressed in all CJ samples (Table S9). Genes that were ex-pressed in MHB but not in CJ included four upregulated genes (pgpA, cmeR, tssJ and macB1) and four downregulated genes (ndk, corC, A0W68_RS03455 (HAD-IB family hydrolase) and A0W68_RS06390 (hypothetical protein)). Two genes were significantly expressed in CJ but not in MHB; these included upregulated corA (magnesium/cobalt transporter) and downregulated A0W68_RS09110 (NYN-domain-containing protein). Table 2. Common genes of C. coli HC2-48 showing significant changes in expression at all incubation times (0 h, 0.5 h, 24 h and 48 h) and in both MHB and CJ media at 4 • C as compared to the control (microaerobic conditions in MHB at 42 • C).

Functional Group Upregulated Genes Downregulated Genes
Energy production/conversion

Functional Group Upregulated Genes Downregulated Genes
Cell motility  Table 3. DEGs in C. jejuni OD2-67 that were significantly expressed in MHB and CJ at 4 • C and at all incubation times (0 h, 0.5 h, 24 h and 48 h) as compared to the control (microaerobic conditions in MHB at 42 • C).

Functional Group Assignments of DEGs
Among DEGs expressed (4 °C vs. 42 °C) at one or more incubation times, 10.83% of C. coli HC2-48 and 25.62% of C. jejuni OD2-67 genes were assigned to the 'unknown function' category ( Figure 2A,B, group S).  The highest percentage of differential expression in C. coli HC2-48 was observed for cell motility (group N), and this group assignment was consistent for all incubation times and medium formulations ( Figure 2C). For C. jejuni OD2-67, genes from energy production/conversion, cell motility, post-translational modification/chaperones and defense mechanisms (groups C, N, O and V, respectively) were differentially expressed at 4 • C in both media ( Figure 2D).
In general, larger fold-change values were observed for upregulated C. coli HC2-48 genes in both media than for downregulated genes; however, exceptions were genes in the following functional groups: N (cell motility), I (lipid transport/metabolism), O (post-translational modification/chaperones) and G (carbohydrate transport/metabolism) (Figures 3 and 4). Accumulative expression values (fold-change) for the genes related to amino acid transport/metabolism (group E) in C. coli were greater for upregulated genes than downregulated genes in MHB, but it was found to be the opposite in CJ. In C. jejuni OD2-67, downregulated genes in functional group J (translation/ribosome structure, O (post-translational modification, chaperones) and D (cell cycle control, cell division, chromosome partitioning) had larger fold-changes values than upregulated genes (Figures 3 and 5). However, accumulative fold-change values of upregulated genes in functional groups C, M, P, H, L, U, F, T, I, V and Q for both media were higher than downregulated genes from respective functional groups (Figures 3 and 5).
mechanisms (groups C, N, O and V, respectively) were differentially expressed at 4 °C in both media ( Figure 2D).
In general, larger fold-change values were observed for upregulated C. coli HC2-48 genes in both media than for downregulated genes; however, exceptions were genes in the following functional groups: N (cell motility), I (lipid transport/metabolism), O (posttranslational modification/chaperones) and G (carbohydrate transport/metabolism) (Figures 3 and 4). Accumulative expression values (fold-change) for the genes related to amino acid transport/metabolism (group E) in C. coli were greater for upregulated genes than downregulated genes in MHB, but it was found to be the opposite in CJ. In C. jejuni OD2-67, downregulated genes in functional group J (translation/ribosome structure, O (posttranslational modification, chaperones) and D (cell cycle control, cell division, chromosome partitioning) had larger fold-changes values than upregulated genes (Figures 3 and  5). However, accumulative fold-change values of upregulated genes in functional groups C, M, P, H, L, U, F, T, I, V and Q for both media were higher than downregulated genes from respective functional groups (Figures 3 and 5). Among the DEGs of C. coli HC2-48 found in both media (CJ and MHB) and at all incubation times, gene enrichment analysis in STRING found significant enrichment (FDR < 0.05) in the KEGG pathway for flagellar assembly (https://www.genome.jp/dbgetbin/www_bget?ko02040, accessed on 9 March 2020). Although C. jejuni OD2-67 lacked a KEGG pathway that was enriched for all incubation times and media, Gene Ontology analysis showed that the ribosome/ribonucleoprotein complex was significantly enriched (FDR < 0.05) for all sampling times in MHB.

Validation of RNA-Seq Data by qRT-PCR
Four DEGs with distinct expression patterns were used to confirm RNA-seq data by qRT-PCR. Two upregulated genes, namely lptF and AR446_RS04795, were chosen to validate RNA-seq results for C. coli HC2-48, and the downregulated genes futA1 and flgN were used to validate results for C. jejuni OD2-67. The expression profiles obtained by qRT-PCR were consistent with results obtained with RNA-seq, indicating that the RNA-seq data are reliable ( Figure 6). Pathogens 2023, 12, x FOR PEER REVIEW 11 of 23 Among the DEGs of C. coli HC2-48 found in both media (CJ and MHB) and at all incubation times, gene enrichment analysis in STRING found significant enrichment (FDR < 0.05) in the KEGG pathway for flagellar assembly (https://www.genome.jp/dbgetbin/www_bget?ko02040, accessed on 9 March 2020). Although C. jejuni OD2-67 lacked a KEGG pathway that was enriched for all incubation times and media, Gene Ontology analysis showed that the ribosome/ribonucleoprotein complex was significantly enriched (FDR < 0.05) for all sampling times in MHB.

Common DEGs in C. coli and C. jejuni (4 • C vs. 42 • C)
Analysis of RNA-seq data indicated that 1386 and 1385 genes from C. coli HC2-48 and C. jejuni OD2-67, respectively, were shared orthologs. However, only 11 genes had fold-change values with FDR < 0.05 at all incubation times and in both media and strains (Table 4 and Table S10), but only two genes, fliS and flgN, were differentially expressed with significant fold change (fold-change values ≥ 1.5 or ≤−1.5 and FDR < 0.05 for both strains at all data points in both media. Pathogens 2023, 12, x FOR PEER REVIEW 12 of 23

Validation of RNA-Seq Data by qRT-PCR
Four DEGs with distinct expression patterns were used to confirm RNA-seq data by qRT-PCR. Two upregulated genes, namely lptF and AR446_RS04795, were chosen to validate RNA-seq results for C. coli HC2-48, and the downregulated genes futA1 and flgN were used to validate results for C. jejuni OD2-67. The expression profiles obtained by qRT-PCR were consistent with results obtained with RNA-seq, indicating that the RNAseq data are reliable ( Figure 6).   . qRT-PCR validation of RNA-seq results. Two genes that were upregulated in RNA-seq data, lptF and AR446_RS04795, were used to follow expression in C. coli HC2-48, and two downregulated genes, futA1 and flgN, were used to validate RNA-seq data for C. jejuni OD2-67. Samples were incubated in MHB or CJ medium at different incubation times (0 h, 0.5 h, 24 h and 48 h), and expression was compared to control strains cultivated in MHB at 42 °C with microaeration. Triplicate biological replicates were used for qRT-PCR analysis, and error bars represent the standard error of means (mean ± SEM).

Common DEGs in C. coli and C. jejuni (4 °C vs. 42 °C)
Analysis of RNA-seq data indicated that 1386 and 1385 genes from C. coli HC2-48 and C. jejuni OD2-67, respectively, were shared orthologs. However, only 11 genes had fold-change values with FDR < 0.05 at all incubation times and in both media and strains (Tables 4 and S10), but only two genes, fliS and flgN, were differentially expressed with significant fold change (fold-change values ≥ 1.5 or ≤−1.5 and FDR < 0.05 for both strains at all data points in both media.  Figure 6. qRT-PCR validation of RNA-seq results. Two genes that were upregulated in RNA-seq data, lptF and AR446_RS04795, were used to follow expression in C. coli HC2-48, and two downregulated genes, futA1 and flgN, were used to validate RNA-seq data for C. jejuni OD2-67. Samples were incubated in MHB or CJ medium at different incubation times (0 h, 0.5 h, 24 h and 48 h), and expression was compared to control strains cultivated in MHB at 42 • C with microaeration. Triplicate biological replicates were used for qRT-PCR analysis, and error bars represent the standard error of means (mean ± SEM).

Influence of Medium Formulation on Gene Expression (CJ vs. MHB)
Although most genes in the two Campylobacter spp. had similar expression patterns (Figure 7), some genes were differentially expressed in the different medium formulations (CJ vs. MHB) (Figure 8). In C. coli HC2-48, 229 DEGs were significantly ex-pressed in CJ vs. MHB at one or more sampling times (Cc48_CJ_0h/Cc48_MHB_0h, Cc48_CJ_0.5h/Cc48_MHB_0.5h, Cc48_CJ_24h/Cc48_MHB_24h and Cc48_CJ_48h/Cc48_ MHB_48h). Among these DEGs, no gene was significantly expressed for all sampling times, but 31 genes had positive fold-change values in CJ vs. MHB (Tables S11 and S12), and 23 genes had negative fold-change values in CJ vs. MHB at all sampling times (Table S13). Among the orthologs, torD (molecular chaperone TorD family protein) was upregulated (at one or more sampling times) for both C. jejuni and C. coli in CJ when compared to MHB at 4 • C. Similarly, katA (catalase) was downregulated (at one or more sampling times) for both species in CJ as compared to MHB at 4 • C.
Prolonged survival of Campylobacter species at lower temperatures is influenced by multiple factors including nutrient availability, environment and inherent characteristics of the strain [1, 2,22,23]. A nutrient-rich environment such as CJ provides a nutritional and protective environment that enhances Campylobacter survival at lower temperatures [1]. In a previous study in our laboratory, both Campylobacter strains (C. coli HC2-48 and C. jejuni OD2-67) used in this study could not produce colonies after two days of incubation in MHB medium at 4 • C but could survive up to fourteen days or more in other tested media (chicken liver juice, beef liver juice, chicken juice and beef juice) at 4 • C [1]. However, Campylobacter strains can also survive for long periods in nutrient-poor environment despite losing cultivability [11]. Transcriptomic analysis of these bacteria during lower temperatures provides insight into their survival mechanisms and is a better approach for studying gene expression in variable environments [24]. Furthermore, changes in the incubation period, medium composition, temperature and atmospheric conditions alters expression in Campylobacter genomes [9,12,13]. In the current study, RNA-seq revealed that a large number of genes in two Campylobacter spp. were impacted by temperature fluctuations (from 42 • C to 4 • C). At 4 • C, medium composition (CJ vs. MHB) and incubation time also altered the Campylobacter transcriptome.
In this study, relatively few orthologous genes were differentially expressed in both Campylobacter spp. when data were compared for temperature, medium formulation and incubation time. For example, our results show that mreB, which is involved in bacterial cell shape, was upregulated for C. coli but downregulated for C. jejuni. Temperature fluctuations are known to differentially impact Campylobacter gene expression; for example, the transcriptomes of C. coli and C. lari showed considerable variability in response to heat stress [15].
The oxidative stress response is reportedly a component of the Campylobacter response to cold shock [10,11,25], and the oxidative stress response genes katA and sodB, were previously upregulated at low temperatures in C. jejuni [25]. A previous study demonstrated that trxB, which encodes thioredoxin-disulfide reductase, had a potential role in the response of C. jejuni to oxidative stress [26]; however, in the current study, trxB was downregulated for both C. jejuni and C. coli in CJ and MHB media at all incubation times. Although msrP was downregulated in C. jejuni OD2-67 at all incubation times and in both media, it was either downregulated or unaltered in C. coli HC2-48. MsrP helps to repair oxidized proteins in the bacterial envelope during oxidative stress [27], and the oxidative stress response during cold shock might vary with the Campylobacter strain, medium and time of incubation. Although temperature fluctuations were closely monitored in this study, the exposure of bacterial samples to atmospheric oxygen during sample processing might cause in discrepancies in the oxidative stress response and gene expression. The iron concentration in media also influences the expression of genes involved in oxidative stress; for example, katA, cj1386, ahpC and trxB were repressed by iron [28]. Likewise, iron ions also reported to play role in the formation of oxygen radicals mediated by Fenton reaction in the bacterial cells [29][30][31][32][33]. Thus, variation in the iron content of CJ might have impacted katA expression in the current study. Interestingly, upregulation of fur (transcriptional repressor) was found in MHB for C. coli HC2-48 {Cc48_MHB_(0.5 h, 24 h, 48 h) vs. Cc48_MHB_0h} and in CJ for C. jejuni OD2-67 {Cj67_CJ_(0.5 h, 24 h, 48 h) vs. Cj67_CJ_0h} and might explain the reduced expression of genes related to oxidative stress and iron acquisition [28,34]. Meanwhile, perR, a transcriptional regulator that also plays role in oxidative stress and iron metabolism, was found upregulated in CJ for C. coli HC2-48 at 0.5 h {for both Cc48_CJ_0.5h/Cc48_42 and Cc48_CJ_0.5h/Cc48_MHB_0.5h) [28,34].
ClpB (AAA family ATPase) functions in various stress responses (oxidative, heat shock, starvation) and virulence in bacteria [2,35]. In our study, clpB was significantly downregulated in both Campylobacter spp. at low temperatures as compared to 42 • C in CJ and MHB. In a previous report, clpB expression in C. jejuni was induced by heat shock and repressed at low temperatures in MHB [13]. It was proposed that heat shock proteins such as ClpB, GroEL, GrpE, HrcA, CbpA, HspR, DnaK and GroES might not be required for survival at lower temperatures [13]. In the current study, these genes were generally downregulated or unaltered for the two Campylobacter strains. Hence, downregulation of heat shock response proteins might be common for Campylobacter spp. at temperatures lower than the optimum for growth.
A number of flagellar genes (flaG, fliS, flgG, flgP, flgN) were downregulated in the two Campylobacter spp. in both CJ and MHB and at all incubation times. A few flagellar genes (e.g., fliH, fliN and fliW) were upregulated or unaltered in all experiments; interestingly, fliK, fliW and fliY were upregulated, but flhB and flgC were downregulated in CJ vs. MHB at one or more incubation times in C. coli HC2-48. For C. jejuni OD2-67, flgM was downregulated in CJ vs. MHB at 0 and 48 h. Downregulation of genes encoding flagellar proteins at suboptimal temperatures was previously reported for C. jejuni and may help the bacterium to conserve energy during adverse environmental conditions [13]. Another study reported upregulation of flg and downregulation of fli genes in C. jejuni at low temperatures [9]. In the current study, a greater number of flagellar genes was impacted in C. coli vs. C. jejuni; furthermore, variability in the expression of flagellar genes might be caused by variation in Campylobacter strains and medium formulations [9,11,13].
Suboptimal temperatures impacted the expression of Campylobacter genes that function in translation and ribosome structure [13]. In the current study, the expression of genes with roles in translation and ribosomal structure/biogenesis was highly represented in both C. coli and C. jejuni. A large proportion of translation/ribosome genes was upregulated in C. coli HC2-48 but downregulated in C. jejuni OD2-67 ( Figure 3). Maintenance of cellular function during cold temperatures is essential and preserves the functionality of translational machinery [13], and the expression of these genes varies among Campylobacter spp.
Genes for energy production and conservation were highly upregulated in both Campylobacter strains in this study; however, genes with significant expression in both strains at all incubation times and all medium formulations were limited. For example, ppa and petC encoding inorganic diphosphatase and cytochrome C1, respectively, were common upregulated genes for C. coli HC2-48 at 4 • C at all incubation times; however, ppa was unaltered, and petC was generally downregulated in C. jejuni OD2-67. Other genes including atpB, ccpA (cytochrome-c peroxidase), hdrC (FAD-binding oxidoreductase), lldP (L-lactate permease), nuoA (NADH quinone oxidoreductase, subunit 3) and nuoM (NADH quinone oxidoreductase, subunit M) were significantly upregulated in C. jejuni OD2-67 and were generally unaltered or significantly upregulated in C. coli HC2-48. These discrepancies show the interspecific variation in the transcriptome of Campylobacter spp. with respect to genes involved in energy production and conversion. In a prior report, the upregulation of lldP and other genes involved in macromolecule transport in C. jejuni was proposed to help the bacterium to acquire cryoprotectants that enhance survival at low temperatures [13]. In this study, DEGs related to amino acid transport and metabolism were less frequent and mostly downregulated. In contrast, genes involved in cell wall/membrane/envelope biogenesis were generally upregulated in the current study, which supports previous reports indicating that modifications in cell membrane structure might function in the cold-shock response [9,11,13].
Multiple genes encoding the type VI secretion system (T6SS) were upregulated in C. jejuni OD2-67 at 4 • C in CJ and MHB at all incubation times. A previous study reported that the T6SS enhanced the oxidative stress response, host colonization and virulence of Campylobacter strains [36,37]. Among the genes related to twin arginine translocation (TAT) system and Sec dependent pathways, tatC was generally upregulated in C. coli HC2-48, whereas secE and secF were upregulated or unaltered, and secG was downregulated or unaltered. Hence, it seems probable that translocation/secretion systems are important in C. coli HC2-48 at 4 • C. Meanwhile, secY was upregulated and secE was downregulated at one or more time points in C. jejuni OD2-67. No significant change in expression was observed for tat genes in C. jejuni OD2-67.
In this study, many Campylobacter genes were differentially expressed in the two medium formulations at 4 • C. However, a previous report using microarrays identified only eight genes with significantly different expression in CJ as compared to brain heart infusion (BHI) medium at 5 • C [12]. This discrepancy might be attributed to the higher sensitivity of RNA-seq as compared to microarray analysis [28]. Despite the high number of DEGs identified in the present study, we did not identify a common gene in C. coli HC2-48 that was differentially expressed in CJ vs. MHB medium at all incubation times. Only one gene A0W68_RS09275 (EexN family lipoprotein) was significantly expressed (downregulated) at all incubation times for C. jejuni OD2-67 and was plasmid-encoded.